The circulatory system is the first organ system to differentiate in the developing embryo. Kaufman, The Atlas of Mouse Development, Academic Press (1992). Embryonic and yolk sac vascular systems take form in an 8.5 day post-coitum (p.c.) mouse embryo, and a day later the heart beats regularly, circulating primitive blood cells, nutrients, and metabolic waste products. Endothelial cells covering blood vessels provide a barrier between blood and other tissues of the embryo. When organs differentiate and begin to perform their specific functions, the phenotypic heterogeneity of endothelial cells increases. Fenestrated vessels, nonfenestrated vessels with tight junctions and sinusoidal vessels are found, for example, in the kidney, brain, and liver, respectively. In addition, endothelial cells perform specific functions in differentiated tissue. For example, such cells take part in several biochemical and physiological events such as blood cell trafficking, blood clotting, hemostasis, ovulation, wound healing, atherosclerosis, and angiogenesis associated with tumor metastasis.
At least five receptor tyrosine kinase genes are expressed in endothelial cells. Of these, the protein products of the FLT1, KDR/FLK-1, and FLT4 genes belong to receptor tyrosine kinase subclass III; whereas Tie and its close relative Tek (Tie-2) form a novel subclass of their own (Terman, et al., Oncogene, 6: 1677-1683 (1991); Terman, et al., Biochem. Biophys. Res. Comm., 187: 1579-1586 (1992); Aprelikova, et al., Cancer Res., 52: 746-748 (1992); De Vries, et al., Science, 255: 989-991 (1992); Pajusola, et al., Cancer Res., 52: 5738-5742 (1992); Sarzani, et al., Biochem. Biophys. Res. Comm., 186: 706-714 (1992); Galland, et al., Oncogene, 8: 1233-1240 (1993); Millauer, et al., Cell, 72: 835-846 (1993); Oelrichs, et al., Oncogene, 8: 11-18 (1993); Schnurch and Risau, Development, 119: 957-968 (1993)).
Both human and mouse Tie cDNAs have been cloned (Partanen, et al., Mol. Cel. Biol., 12: 1698-1707 (1992); Korhonen, et al., Blood, 80: 2548-2555 (1992); Korhonen, et al., Oncogene, 8: 395-403 (1994); Iwama, et al., Biochem. Biophys. Res. Comm., 195: 301-309 (1993); Sato, et al., Proc. Natl. Acad. Sci. USA., 90: 9355-9358 (1993)). Tie and homologous genes have been isolated from bovine and rat sources (Maisonpierre, et al., Oncogene, 8: 1631-1637 (1993); Sato, et al., Proc. Natl. Acad. Sci. USA., 90: 9355-9358 (1993)).
The 4.4 kb Tie-encoding mRNA encodes a 125 kDa transmembrane protein which is N-glycosylated. In its extracellular domain Tie contains two immunoglobulin-like loops and three epidermal growth factor and fibronectin type III homology regions, which are followed by trans- and juxtamembrane domains connected to a tyrosine kinase domain which is split by a short kinase insert sequence and a carboxyl terminal tail (Partanen, et al., Mol. Cel. Biol., 12: 1698-1707 (1992); Korhonen, et al., Oncogene, 8: 395-403 (1994); Sato, et al., Proc. Natl. Acad. Sci. USA., 90: 9355-9358 (1993)). Both Tie and TEK have been localized to mouse chromosome 4 at a distance of 12.2 centimorgans from each other. Such receptors are uniformly expressed in endothelial cells of various blood vessels during embryonic development, although the expression of Tek mRNA appears to begin 0.5 days earlier than the expression of Tie. In adult mice, the expression of Tie mRNA persists in vessels of the lung whereas in the heart and brain it appears to decrease. Korhonen, et al., Oncogene, 8: 395-403 (1994). Production of Tie mRNA is enhanced during ovulation and wound healing and in human glioblastomas (Korhonen, et al., Blood, 80: 2548-2555 (1992)).
Endothelial cells play a key role in gene therapy directed to diseases involving endothelial cells and blood vessels, such as establishment of neovascularization or inhibition of angiogenesis, and control of inflammatory trafficking of leukocytes. One approach to the treatment of vascular disease is to express genes at specific sites in the circulation that might ameliorate the disease in situ. Because endothelial cells are found at diseased sites, they represent logical carriers to convey therapeutic agents that might include anticoagulant, vasodilator, angiogenic or growth factors. Accordingly, the genetic modification of endothelial cells represents a therapeutic approach to the treatment of many vascular disorders, including hypertension, atherosclerosis and restenosis. For example, endothelial cells expressing growth inhibitory proteins could be introduced via catheter to the angioplasty site to prevent local intimal hyperplasia and clinical restenosis. The luminal surface of vascular grafts could also be lined with genetically modified endothelial cells producing therapeutic proteins which prevent thrombosis or promote repopulation (Nabel, et al., J. Am. Coll. Cardiol., 17: 189B-194B (1991)).
Endothelial cells lining blood vessels are easily transfected with methods using liposomes, adenovirus vectors and retroviral vectors (Nabel, et al., J. Am. Coll. Cardiol. 17: 189B-94B). Endothelial cells are also in direct contact with blood and are therefore optimal sources for production and secretion of desired proteins or peptides into the blood stream. For example, the Factor VIII gene may be introduced into endothelial cells under an endothelial-cell-specific promoter, resulting in correction of hemophilia if the protein were expressed in sufficeint quantity. On the other hand, endothelial cells are also useful for delivery of peptides or proteins expressed in them into tissues. In this regard, a selective expression of a particular gene regulatory element in endothelial cells of the microvasculature (capillaries) is extremely useful, given that most of the cell surface area facing the vascular lumen consists of microvascular endothelial cells.
Control elements of the endothelial-cell-specific promoters may be further subdivided and dissected into functional elements and units according to methods standard in the art. The Tie protein is expressed in certain endothelial cells and in a small fraction of human bone marrow cells including hematopoietic progenitor cells. See Batard et al., Blood, 68:1729-1735 (1996). Therefore, it is likely that the Tie promoter is active also in some hematopoietic cells. However, expression of the Tie promoter in hematopoietic cells may be controlled by elements which are distinguishable from endothelial-cell-specific elements and may be dissected away while retaining the endothelial cell specificity of the promoter.
The present invention provides a novel promoter associated with the gene encoding the Tie receptor tyrosine kinase for use in therapeutic and diagnostic procedures. In addition, the promoter may prove useful in the production of desired proteins to the blood or tissues of animals.